Pyk2-dependent phosphorylation of LSR enhances localization of LSR and tricellulin at tricellular tight junctions

Tight junctions (TJs) are cellular junctions within the mammalian epithelial cell sheet that function as a physical barrier to molecular transport within the intercellular space. Dysregulation of TJs leads to various diseases. Tricellular TJs (tTJs), specialized structural variants of TJs, are formed by multiple transmembrane proteins (e.g., lipolysis-stimulated lipoprotein receptor [LSR] and tricellulin) within tricellular contacts in the mammalian epithelial cell sheet. However, the mechanism for recruiting LSR and tricellulin to tTJs is largely unknown. Previous studies have identified that tyrphostin 9, the dual inhibitor of Pyk2 (a nonreceptor tyrosine kinase) and receptor tyrosine kinase platelet-derived growth factor receptor (PDGFR), suppresses LSR and tricellulin recruitment to tTJs in EpH4 (a mouse mammary epithelial cell line) cells. In this study, we investigated the effect of Pyk2 inhibition on LSR and tricellulin localization to tTJs. Pyk2 inactivation by its specific inhibitor or repression by RNAi inhibited the localization of LSR and downstream tricellulin to tTJs without changing their expression level in EpH4 cells. Pyk2-dependent changes in subcellular LSR and tricellulin localization were independent of c-Jun N-terminal kinase (JNK) activation and expression. Additionally, Pyk2-dependent LSR phosphorylation at Tyr-237 was required for LSR and tricellulin localization to tTJs and decreased epithelial barrier function. Our findings indicated a novel mechanism by which Pyk2 regulates tTJ assembly and epithelial barrier function in the mammalian epithelial cell sheet.

紧密连接（Tight junctions，TJs）是存在于哺乳动物上皮细胞层中的细胞连接，可作为物理屏障阻断分子在细胞间隙内的转运。紧密连接失调可引发多种疾病。三细胞紧密连接（tricellular TJs，tTJs）作为紧密连接的特化结构变体，由哺乳动物上皮细胞层三细胞接触位点处的多种跨膜蛋白（如脂解刺激脂蛋白受体[lipolysis-stimulated lipoprotein receptor，LSR]和三细胞蛋白[tricellulin]）构成。然而，将LSR与三细胞蛋白招募至三细胞紧密连接的分子机制尚未完全阐明。既往研究显示，酪氨酸磷酸化抑制剂9（tyrphostin 9）作为富脯氨酸酪氨酸激酶2（Pyk2，一种非受体酪氨酸激酶）与受体酪氨酸激酶血小板衍生生长因子受体（platelet-derived growth factor receptor，PDGFR）的双重抑制剂，可在EpH4细胞系（一种小鼠乳腺上皮细胞系）中抑制LSR与三细胞蛋白向三细胞紧密连接的招募。本研究探讨了Pyk2抑制对LSR与三细胞蛋白在三细胞紧密连接处定位的影响。通过特异性抑制剂使Pyk2失活，或通过RNA干扰（RNAi）抑制Pyk2表达，均可在不改变EpH4细胞中二者表达水平的前提下，阻碍LSR与下游三细胞蛋白向三细胞紧密连接的定位。Pyk2介导的LSR与三细胞蛋白的亚细胞定位变化，并不依赖c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）的激活与表达。此外，Pyk2介导的LSR在酪氨酸237（Tyr-237）位点的磷酸化，是LSR与三细胞蛋白定位于三细胞紧密连接以及维持上皮屏障功能的必要条件，该磷酸化事件还会削弱上皮屏障功能。本研究揭示了Pyk2调控哺乳动物上皮细胞层中三细胞紧密连接组装与上皮屏障功能的全新机制。